Method for increasing the production of starch-hydrolyzing enzymes



nite tes METHOD FOR INCREASING THE PRODUCTION OF STARCH-HYDROLYZING ENZYMES Koichi Yamada, RAM floncho dori, Nakano-ku, Tokyo, Japan My invention relates to a method of producing improved yields of starch-hydrolyzing enzymes by the fermentation of nutrient media, and more particularly, it relates to the production of starch-hydrolyzing enzymes by the fermentation of saccharides in the presence of phytic acid or salts thereof.

The production of starch-hydrolyzing enzymes has been accomplished in the past by the fermentation of starch-containing nutrient media containing different forms of nitrogen sources and, as needed, small amounts of inorganic salts using either surface culture or submerged culture methods. For example, strains of the genus Aspergillus have been cultured in media containing starch and a nitrogen source, such as nitrite, nitrate, aspartic acid, asparagine or urea, and a small quantity of inorganic salts. Occasionally, vitamins, steroids, hormones, amino acids, organic acids or inorganic acids have been added to culture media, such as the above, for increasing the yields of the starch-hydrolyzing enzymes.

I have now discovered that the yields of starch-hydrolyzing enzymes obtainable by previously known methods can be materially increased by including in the fermentation media a small quantity of phytic acid or the salts thereof.

My improved process for the production of starchhydrolyzing enzymes has the added advantage of permitting the substitution of various saccharides for the starch used in the prior art processes. 1 have discovered that even in media not containing starch, very potent starch-hydrolyzing enzymes can be produced in a short time if the fermentation is carried out under conventional methods in the presence of phytic acid or salts thereof using as a source of carbon a variety of saccharides such as the monosaccharides, glucose, fructose, mannose, galactose, etc.; and disaccharides, such as maltose, sucrose, lactose, etc.; and that even in media containing starch as the carbon source the production of starchhydrolyzing enzymes is markedly augmented.

In carrying out my process for the production of starch-hydrolyzing enzymes, I can use any of the starchcontaining culture media of the prior art, or similar culture media in which the starch has been substituted in whole or in part by a saccharide or mixture of saccharides. If starches are used as the source of the carbon, it can be used in either the form of pure or crude starches of any conventional source, such as grains or potatoes;

or in place of the pure starches, I can use starch-containing materials such as potatoes or cereals in suitably comminuted form. Suitable cereals include maize, wheat, oats, etc. Similarly, I can use either a pure saccharide of the type set forth above or a saccharide-containing material, such as, for example, beet or cane- It is generally necessary, however, to add' sodium nitrate and the like; organic nitrogen sources such as cornsteep liquor, urea, etc.

I have now discovered that by adding to either the cul ture media of the prior art containing starch as the sole carbon source, or similar media in which a saccharide is substituted in part or in whole for the starch, an amount of phytic acid or a salt thereof in an amount ranging from 0.01% to 0.1%, the yields of starch-hydrolyzing enzymes are markedly increased over any obtainable by any prior art method. When used in the salt form, I can use any non-toxic salt of phytic acid. For most purposes,

however, I have found it preferable to use salts such as sodium phytate, potassium phytate, calcium phytate, barium phytate, magnesium phytate, and the like.

The culture media used in my process is prepared by mixing the various ingredients thereof, including the phytic acid or salt thereof, in any conventional manner, followed by sterilization with steam under pressure, using conventional methods. The sterilized medium so obtained is then inoculated with a starch-hydrolyzing filamentous fungus such as for example, Aspergillus oryzae, Aspergillus awamori, or Rhizopus javanicus or with starch-hydrolyzing producing bacilli, such as, for example, Bacillus subtilis, and then cultivated under conventional methods, such as shaking, aeration, or agitating aeration at temperatures of the order of 30 C. in the case of fungi, and of the order of 37 C. in the case of bacteria. A notable growth of mycelia appears in about two days time after inoculation and the production of starch-hy drolyzing enzymes reaches a maximum within three to four days time after inoculation.

The starch-hydrolyzing enzymes so produced are recovered by any conventional method.

The enzymes produced in accordance with my invention exhibit dextrinogenic amylase and saccharogenic amylase activities in high concentrations. The potency of the dextrinogenic amylase and saccharogenic amylase can be estimated in the following manner.

I. QUANTITATIVE ESTIMATION OF DETRINOGENIC AMYLASE Place 20 ml. of 2% soluble starch solution into a ml. flask and add 2 ml. of acetate bufier solution (pH 4.8). Next add 2 m1. of a solution containing the starchhydrolyzing enzyme (dextrinogenic amylase) at 30 (3., mix the ingredients and calculate the time (expressed in minutes) required for the iodine reaction to disappear. The higher the potency, the lower the numerical value.

n. QUANTITATIVE ESTIMATION OF SACCHAROGENIC AMYLASE a 20.m1.-.9f2.% sol b t rc 8911115011 into a 0 ml. flask and add 2 ml. of acetate buffer 'selun'oatprr 4.8). Then add 2 ml. of a solution containing the saccharogenic amylase at 30 C. to the mixture so obtained. To estimate quantitatively reducing sugar formed by the saccharogenic amylase in the mixture, a sample of the solution is immediately taken and analyzed by Somoygis method. Additional samples are taken at thirty minute intervals andsimilarly analyzed. From the value so obtained, the potency of saccharogenic amylase is determined by the following formula: Potency of sacoharogenie amylase reduced sugar produced (mg) total sugar (mg) in the mixture In this case, the higher the numerical value, the greater the potency.

The followingexamples will serve to illustrate my invention. It is understood, however, that I do not intend to be limited to the components, temperatures, proportions', e'tc., set forth, but rather it is intended that all equivalents obvious to those skilled in the art be included within the scope of my invention.

Example II Media were prepared using starch instead of mono- Example I saccharides as the carbon source in the medium of Ex- A di i i g a monosaccharide (24% 5 ample 1. Three k1nds of media, 1.e., one containmg no monium nitrate (0.5%), disodium hydrogen phosphate addltlonal Substance, I1e C 11ta1n1ng a d P Y' acid (0.1%),potassium chloride (0.05%),and magnesium suland one contalmng added {I10S1t01 (0.01%) fat was prepared To one series f 5 were prepared. Each of the three media was inoculated Erlenmeyer flasks each containing 100 ml. of this medium wlth Asperglllus f R 337 and cultured in the l i phytate 001% was added To other series same manner as described in Example I. The results are of flasks containing the medium, no calcium phytate was Shown 111 Table H belowadded.

The pH of each series of flasks was then adjusted to 5.0 with hydrochloric acid. After sterilization for TABLE I1 minutes with steam at a pressure of 15 lbs, and cooling, 15 each flask was inoculated with a very small amountof 11mm Dexmnm Sacchawmmmms spores of Aspergzllus nzger NRRL 337 and fermentation q genlic genlic of mi permitted to take place at a temperature of about 30" Medium gggf pH ,33 9," C. on a shake table operating at a speed of about 240 broth r.p.m. One gram of sterilized calcium carbonate was added to each flask 24 hours after inoculation, or as 40 Q needed, to keep the pH of the fermenting medium within Basalmedmm g; 2;; gig 15 5 the range 4-7. Starch-hydroly'zing enzymes having the Basalmemum 40 -0 .3 70 +Inoslhlghest potency were obtained after 1ncubat1on for 3-4 tol. g; :2 8 3 days Basal medium g Results obtained with four monosacchandes. with and +Phytie acid. 88 5 77 without added calcium phytate, are shown in Table I.

TABLE I Demim' saccham' As is clearly shown in the above table starch-hydroly- Monosaccharides Calcium tion pH genie genie used Phytato Period, Amylase Amylase zlng enzymes are produced in increased quantities only Days Activity Activity when phytic acid is added and not by inositol, which is related to phytic acid. l g 3 Not added- 4 5 30 g Example III S 238 "i 1% Media were prepared as described in Example II using Added 3 5 starch as the carbon source, and 0.01% phytic acid. The

4 7.0 4 18.9 G1 5 4 flasks were then inoculated with three strains of molds; ucose g-g 382 g-% namely, Aspergillus oryzae, Aspergillus awamori, and added" 4 Rhizopus javanicus, and culturing carried out in the same 5 manner as described in Example I. The results obtained 2 6.0 30 2.1 Add d 3 9 are shown m Table III below. e t it 2 2a Fructose 2 5 3o Examp 2 IV NOt added 2 232 282 83.3 A medium consisting of starch (25%), disodium byg-g 8 drogen-phosphate (0.1% calcium chloride (0.05% and Added 3 magnesium nitrate (0.05%) was prepared as described 3-8 2 3-2 in the preceding example. However, as the nitrogen Mamwse 2 5 source, 0.5% each of yeast extract, peptone, and dis- Not added" 2 g-g g8 8 tillers dried solubles was substituted for the ammonium 5 2 nitrate in each medium. Culturing was carried out in g %g the same manner described in Example I, using Aspergil- Galactose Added i 4 5:5 19 2253 his niger NRRL 337.

5 5.5 a0 1.2 The above media were compared with a medium containing ammonium ntirate (0.5 as the nitrogen source.

TABLE III Ineuba- Dextrino- Bacchuro- Milligrams tion genie genie of Sugar Microorganisms Phytio Acid Period, pH Amylase, Amylase, Mycellum Consumed Days Activity Activity per 10 ml. (percent) of Broth not 2 5.0 18 1.7 45 31.2 3 5.5 28 3.4 45 43.7 1189mm 2 6.0 2 21.1 08.0 2 z-a .2 as 22-1 not added.--

g s3 3% 99 not added-.-

2 5.0 32 0.4 50 70.3 Rhizopusjavanfeua added. 2 M) 32 0 32 7&8 3 5.0 33 2.6 90.0

5 Phytic acid (0.05%) was added as indicated. The results are shown in Table IV below.

4. The process of claim 1 wherein the nutrient me, dium contains glucose as a source of carbon.

TABLE IV Incuba- Dextriuosaccharo- Source of Nitrogen Phytie Acid tion pH genie genie Period, Amylase, Amylase, Days Activity Activity 2 5. 5 30 1. 21 Ammonium Nitrate Not added... 2 g 5.0 30 2 5. 2 4 25. 3 D0 n iii 5 a:

5 5. 5 2 7. 5 30 1. 1 Yeast Extract Not added 2 5 7. 5 9 4. 7 2 6. 5 30 1. 3

Pep

Z 2;? a: 5 5. 5 14 5.2 2 5. 5 16 5. 6 Distillers Dried Solubles d0 i g 5 5. 5 9 9. 3

From the above table, it is clear that the addition of a trace of phytic acid to a medium containing a simple inorganic salt such as ammonium nitrate as .the nitrogen source leads to more advantageous production of starchhydrolyzing enzymes than the .use of some organic nitrogen sources, and therefore, that the use of phytic acid is economically of high efiiciency.

Now having described my invention, what I claim is:

1. In a process for the production of starch-hydrolyzing enzymes selected from the group consisting of dextrinogenic amylase and saccharogenic amylase by the fermentation of a nutrient medium with an enzyme-producing microorganism selected from the group consisting of dextrinogenic amylase-producing microorganisms and saccharogenic amylase-producing microorganisms, the improvement which consists of incorporating in said nutrient medium a material selected from the group consisting of phytic acid and non toxic salts of phytic acid in amounts sufficient to improve the yields of said starch-hydrolyzing enzymes.

2. The process of claim 1 wherein the nutrient medium contains a source of carbon selected from the group consisting of monosaccharides and disaccharides,

3. The process of claim 1 wherein the nutrient medium contains starch as a carbon source.

5. The process of claim 1 wherein the fermentation is carried out at a temperature of approximately 30 C. for 2-4 days using an enzyme-producing filamentous fungi selected from the group consisting of dextrinogenic amylase-producing filamentous fungi and saccharogenicamylase-producing filamentous fungi.

6. The process of claim 1 wherein the fermentation is carried out at a temperature of about 37 C. for 1-3 days time using an enzyme-producing bacterium selected firom the group consisting of dextrinogenic amylase-producing bacteria and saccharogenic amylase-producing bacteria.

7. The process of claim 1 wherein the fermentation is carried out with a mold selected from the group consisting of Aspergillus niger, Aspergillus oryzae, Aspergillus awamori, and Rhizopus javanicus.

8, The process of claim 1 wherein from 0.01% to 0.1% of a material selected from the group consisting of phytic acid and nontoxic salts of phytic acid are included in said medium.

References Cited in the file of this patent Proc. Int, Symposium on Enzyme Chemistry, Tokyo and Kyoto, 1957, Weda, pages 491 to 494; Okazaki, pages 494 to 499. 

1. IN A PROCESS FOR THE PRODUCTION OF STARCH-HYDROLYZING ENZYMES SELECTED FROM THE GROUP CONSITING OF DEXTRINOGENIC AMYLASE AND SACCHAROGENIC AMYLASE BY THE FERMENTATION OF A NUTRIENT MEDIUM WITH AN ENZYME-PRODUCING MICROORGANISM SELECTED FROM THE GROUP CONSISTING OD DEXTRINOGENIC AMYLASE-PRODUCING MICOORGANISMS AND SACCHAROGNEIC AMYLASE-PRODUCING MICROORGANISMS, THE IMPROVEMENT WHICH CONSISTS OF IMCORPORATING IN SAID NUTRIENT MEDIUM A MATERIAL SELECTED FROM THE GROUP CONSISTING OF PHYTIC ACID AND NON-TOXIC SALTS OF PHYTIC ACID IN AMOUNTS SUFFICIENT TO IMPROVE THE YIELDS OF SAID STARCH-HYDROLYZING ENZYMES. 